PROJECT SUMMARY F1Fo ATP synthase is a fundamental energy conversion complex found in all known cells. This complex uses an electrochemical gradient of ions to drive a rotary mechanism that results in the synthesis of adenosine triphosphate (ATP), the universal chemical energy currency of life. In E. coli, the proton-driven Fo motor, embedded in the cell membrane, consists of a decameric subunit c rotor ring in contact with subunit a of the stator. Protons move through Fo via two offset aqueous half channels. Protons enter through subunit a from the periplasm (P-side) to the c-ring. After c-ring rotation, protons exit to the cytoplasm (N-side) by a half channel formed at the a-c interface. Clusters of residues along the N-side channel have been implicated in proton translocation and a possible gating mechanism, but the roles of these residues are unclear. This project seeks to engage undergraduate student researchers in a combined biochemical and biophysical approach to characterize interactions between the rotor and stator and establish their role(s) in proton transport. Aim 1 will systematically examine mutations and chemical modifications of several key residues on the N-side of the a-c interface with a battery of in vitro biochemical assays to determine the role of each residue in proton-driven ATP synthesis and ATP-driven proton pumping. Aim 2 will use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to probe the structure and structural dynamics of the loop regions of subunit a that interact with the c-ring. Patterns of spin label mobility, accessibility, and proximity will reveal whether interacting cytoplasmic loops at the a-c interface undergo conformational changes during ATP synthesis or hydrolysis. Together, the structural and functional data will contribute to understanding the molecular mechanism of proton transport in this important biomolecular machine and inform future drug development.